For DNA methylation analysis on the whole genome, MagMeDIP kit can be coupled with Next-Generation Sequencing. To perform MeDIP-sequencing we recommend the following strategy:
- Choose a library preparation solution which is compatible with the starting amount of DNA you are planning to use (from 10 ng to 1 μg). It can be a home-made solution or a commercial one.
- Choose the indexing system that fits your needs considering the following features:
- Single-indexing, combinatorial dual-indexing or unique dual-indexing
- Number of barcodes
- Full-length adaptors containing the barcodes or barcoding at the final amplification step
- Presence / absence of Unique Molecular Identifiers (for PCR duplicates removal)
- Standard library preparation protocols are compatible with double-stranded DNA only, therefore the first steps of the library preparation (end repair, A-tailing, adaptor ligation and clean-up) will have to be performed on sheared DNA, before the IP.
CAUTION: As the immunoprecipitation step occurs at the middle of the library preparation workflow, single-tube solutions for library preparation are usually not compatible with MeDIP-sequencing.
- For DNA isolation after the IP, we recommend using the IPure kit v2 (available separately, Cat. No. C03010014) instead of DNA isolation Buffer.
- Perform library amplification after the DNA isolation following the standard protocol of the chosen library preparation solution.
Example of results
Figure 1. qPCR analysis of external spike-in DNA controls (methylated and unmethylated) after IP. Samples were prepared using 1μg – 100ng -10ng sheared human gDNA with the MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after IP has been performed with IPure kit V2 (Diagenode).
Figure 2. Saturation analysis. Clean reads were aligned to the human genome (hg19) using Burrows-Wheeler aligner (BWA) algorithm after which duplicated and unmapped reads were removed resulting in a mapping efficiency >98% for all samples. Quality and validity check of the mapped MeDIP-seq data was performed using MEDIPS R package. Saturation plots show that all sets of reads have sufficient complexity and depth to saturate the coverage profile of the reference genome and that this is reproducible between replicates and repetitive experiments (data shown for 50 ng gDNA input: left panel = replicate a, right panel = replicate b).
Figure 3. Sequencing profiles of MeDIP-seq libraries prepared from different starting amounts of sheared gDNA on the positive and negative methylated control regions. MeDIP-seq libraries were prepared from decreasing starting amounts of gDNA (1 μg (green), 50 ng (red), and 10ng (blue)) originating from human blood with the MagMeDIP kit (Diagenode) and a commercially available library prep kit. DNA isolation after IP has been performed with IPure kit V2 (Diagenode). IP and corresponding INPUT samples were sequenced on Illumina NovaSeq SP with 2x50 PE reads. The reads were mapped to the human genome (hg19) with bwa and the alignments were loaded into IGV (the tracks use an identical scale). The top IGV figure shows the TSH2B (also known as H2BC1) gene (marked by blue boxes in the bottom track) and its surroundings. The TSH2B gene is coding for a histone variant that does not occur in blood cells, and it is known to be silenced by methylation. Accordingly, we see a high coverage in the vicinity of this gene. The bottom IGV figure shows the GADPH locus (marked by blue boxes in the bottom track) and its surroundings. The GADPH gene is a highly active transcription region and should not be methylated, resulting in no reads accumulation following MeDIP-seq experiment.